CN114634497A - 一种半胱氨酸/高半胱氨酸响应的aie荧光探针及其制备方法与应用 - Google Patents
一种半胱氨酸/高半胱氨酸响应的aie荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于荧光探针领域,具体涉及一种半胱氨酸/高半胱氨酸响应的 AIE荧光探针及其制备方法与应用。
背景技术
生物硫醇,主要包括半胱氨酸(Cys)、高半胱氨酸(Hcy)和谷胱甘肽 (GSH),参与生物体内多种生理过程与病理过程。Cys浓度异常可引起许多疾病,如儿童生长缓慢和肝脏病。Hcy的水平与冠心病、皮肤病和阿尔茨海默病密切相关。GSH水平的波动被认为是糖尿病的标志。但是,由于在结构和反应性上极为相似,Cys、Hcy和GSH往往难以区分。
现有技术中,生物硫醇荧光探针都有聚集引起的淬灭(ACQ)效应。荧光团在有机溶剂中具有很强的荧光,但随着溶剂中水的比例不断上升,荧光会逐渐减弱甚至熄灭。2001年,唐本忠课题组首次报道了聚集诱导发射(AIE)效应。与ACQ效应相反,AIE分子在分散状态下几乎不出现荧光,但在聚集状态下会发出强烈的荧光。
目前,小分子荧光探针是检测生物硫醇的主流方法,具有高选择性、高灵敏度和低成本的优点。但传统的ACQ荧光探针的应用生物体内水的比例含量较高而受到极大限制。因此设计开发AIE荧光探针对生物硫醇的检测具有重要的意义。
发明内容
发明目的:针对现有技术中存在的不足,本发明提供了一种生物硫醇选择性好、灵敏度高、聚集引起的淬灭效应不受限制的半胱氨酸/高半胱氨酸响应的AIE荧光探针;
本发明还提供了半胱氨酸/高半胱氨酸响应的AIE荧光探针的制备方法和应用。
技术方案:为了实现上述目的,本发明所述一种半胱氨酸/高半胱氨酸响应的AIE荧光探针,简记为SQM-NBD,其结构式如下式I所示:
上述半胱氨酸/高半胱氨酸是指半胱氨酸或高半胱氨酸。
本发明所述半胱氨酸/高半胱氨酸响应的AIE荧光探针的制备方法,包括如下步骤:
(1)将2-甲基喹啉和碘乙烷溶解于无水乙腈中,在惰性气体氮气保护下回流反应,将反应混合物冷却至室温后,将沉淀物减压抽滤,洗涤,真空干燥后得到化合物1;
(2)将化合物1与丙二腈溶解于乙醇中,然后加入乙醇钠反应,反应结束后,沉淀物减压抽滤,洗涤,真空干燥后得到化合物2;
(3)将4-羟基苯硼酸和5-溴-2-噻吩甲醛和四(三苯基膦)钯溶解在无水四氢呋喃中,加入碳酸钾水溶液,在惰性气体氮气保护下回流反应,反应结束后,将反应混合物冷却至室温,萃取,减压蒸除溶剂,纯化后得到化合物3;
(4)将化合物2和化合物3溶解于无水乙腈中,再加入哌啶,在惰性气体氮气保护下回流反应,将反应混合物冷却至室温,减压蒸除溶剂,纯化后得到化合物SQM-OH。
(5)将SQM-OH与4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)溶解于无水二氯甲烷中,再加入三乙胺,在惰性气体氮气保护下回流反应,反应结束后,将溶剂从混合物中蒸发,残余物纯化得到荧光探针SQM-NBD。
作为优选,步骤(1)将2-甲基喹啉和碘乙烷溶解在无水乙腈中,在惰性气体氮气保护下于85℃下反应回流12h,待反应结束后,将反应混合物冷却至室温后析出大量固体,减压抽滤去溶剂,得到滤饼用冷乙腈洗涤,真空干燥后,得到淡黄色固体,即化合物1。
作为优选,步骤(2)将化合物1和丙二腈溶解于无水乙醇,在-2℃下持续搅拌,然后加入乙醇钠,在-2℃下继续反应0.5h,转至室温继续搅拌反应4h,减压抽滤除去多余溶剂,滤饼用冷乙醇洗涤,真空干燥,得到黄色固体,即化合物2。
作为优选,步骤(3)将4-羟基苯硼酸、5-溴-2-噻吩甲醛和四(三苯基膦) 钯溶解在无水四氢呋喃中,随后快速加入碳酸钾水溶液,在70℃、惰性气体氮气保护下搅拌回流6h,反应结束后,将反应混合物冷却至室温,二氯甲烷萃取,减压蒸除溶剂,通过柱层析纯化得化合物3。
作为优选,步骤(4)将化合物2和化合物3溶解在无水乙腈中,加入哌啶,在85℃、惰性气体氮气保护下搅拌回流8h,将反应混合物冷却至室温后,减压蒸除溶剂,残余物通过柱层析纯化得到化合物SQM-OH。
作为优选,步骤(5)将SQM-OH与NBD-Cl溶解于无水二氯甲烷中,加入三乙胺,在25℃、惰性气体氮气保护下反应回流12h,反应结束后,减压蒸除溶剂,通过柱层析纯化得到荧光探针SQM-NBD。
作为优选,其反应路线如下所示:
本发明所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针在半胱氨酸/高半胱氨酸的响应性检测和在细胞内源性外源性半胱氨酸/高半胱氨酸的成像中的应用。
作为优选,所述响应性检测的过程为:向含有AIE荧光探针的反应体系中分别加入半胱氨酸、高半胱氨酸和谷胱甘肽,将该反应溶液混合均匀后于37℃下孵育,孵育后测定紫外吸收和荧光发射光谱。
作为优选,所述成像的过程为:将Hela细胞于激光共聚焦皿中培养,将荧光探针加入皿中共孵育,用激光共聚焦显微镜进行成像。
有益效果:与现有技术相比,本发明具有如下优点:
(1)该荧光探针的合成简单,使用方便,与半胱氨酸/高半胱氨酸响应后释放出荧光团SQM-OH,与此同时还生了新的荧光物质Cys/Hcy-NBD。
(2)从响应前后的荧光强度变化就可以快速检测半胱氨酸/高半胱氨酸,实现了快速检测的目的。
(3)该荧光探针本身不具有荧光,但可与半胱氨酸/高半胱氨酸快速反应之后,产生明显的荧光信号增强,从而实现对生物硫醇选择性检测。因此,本发明中的荧光探针可以作为一个有效的工具来检测生物硫醇中的半胱氨酸/高半胱氨酸。
附图说明
图1为本发明荧光探针SQM-NBD的高分辨质谱图;
图2(A)为本发明荧光探针SQM-NBD在DMSO-PBS混合溶液中分别与半胱氨酸(Cys)、高半胱氨酸(Hcy),和谷胱甘肽(GSH)孵育后的荧光发射光谱,(B)为荧光探针SQM-NBD在DMSO-PBS混合溶液与半胱氨酸(Cys) 和谷胱甘肽(GSH)共孵育后的荧光发射光谱;
图3为本发明荧光探针SQM-NBD在Hela细胞中的成像图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明中使用的实验方法无特殊说明,均为常规方法。实验所用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中所选用的以下所有试剂皆为市售分析纯或化学纯。
实施例1
半胱氨酸/高半胱氨酸响应的AIE荧光探针的制备方法:
(1)化合物1的合成:将2-甲基喹啉(1.33mL,10.0mmol)和碘乙烷 (1.20mL,15.0mmol)溶解在无水乙腈(15mL)中,并在85℃、惰性气体N2保护下反应回流12h。反应结束后,将反应混合物冷却至室温,析出大量固体,减压抽滤多余溶剂,得到滤饼用冷乙腈洗涤(3×15mL)。干燥后,得到淡黄色固体,即化合物1。
(2)化合物2的合成:将化合物1(0.3g,1mmol)和丙二腈(0.247g, 3.7mmol)溶于无水乙醇(25mL),在-2℃下持续搅拌,然后加入乙醇钠 (0.307g,3.7mmol),在-2℃下继续反应0.5h后,转至室温继续搅拌反应4h,反应完成后,沉淀物减压抽滤除去溶剂,滤饼用冷乙醇洗涤(3×15mL),真空干燥,得到黄色固体,即化合物2。
(3)化合物3的合成:将4-羟基苯硼酸(2.7g,20mmol)、5-溴-2-噻吩甲醛(2.38mL,20mmol)和四(三苯基膦)钯(0.2g,0.2mmol)溶解在无水四氢呋喃(150mL)中,随后快速加入体积分数为22%的碳酸钾水溶液 (40mL),在70℃、惰性气体N2保护下搅拌回流6h,反应结束后,将反应混合物冷却至室温,用二氯甲烷萃取后,减压蒸除溶剂,通过柱层析纯化(体积比为石油醚:乙酸乙酯=5:1)得化合物3。
1H NMR(600MHz,DMSO)δ10.25(s,1H),9.87(s,1H),8.00(d,J=4.0Hz,1H),7.64(d,J= 8.6Hz,2H),7.56(d,J=4.0Hz,1H),6.92(d,J=8.6Hz,2H).
(4)SQM-OH的合成:将化合物2(0.47g,2.0mmol)和化合物3(0.41 g,2.1mmol)溶解在无水乙腈(30mL)中,加入哌啶(0.25mL),在85℃、惰性气体N2保护下反应回流8h,将反应混合物冷却至室温后,减压蒸除多余溶剂,通过柱层析纯化(体积比为二氯甲烷:甲醇=80:1)得到SQM-OH粉末。
1H NMR(600MHz,DMSO)δ9.86(s,1H),8.90(d,J=8.3Hz,1H),8.07(d,J=8.7Hz,1H), 7.91(t,J=7.5Hz,1H),7.64(d,J=15.4Hz,1H),7.62–7.58(m,1H),7.55(d,J=8.1Hz,3H),7.39 (d,J=3.3Hz,1H),7.10(d,J=15.4Hz,1H),7.01(s,1H),6.84(d,J=8.3Hz,2H),4.54(d,J=6.8Hz, 2H),1.42(t,J=6.8Hz,3H).
(5)SQM-NBD的合成:将SQM-OH(0.33g,0.81mmol)与4-氯-7-硝基 -2,1,3-苯并氧杂噁二唑(NBD-Cl)(0.15g,0.75mmol)溶解于无水二氯甲烷 (30mL)中,加入三乙胺(0.15mL),在25℃、惰性气体N2保护下反应回流 12h,反应结束后,减压蒸除溶剂,残余物通过柱层析(体积比为二氯甲烷:甲醇=80:1)纯化得到荧光探针SQM-NBD。
1H NMR(600MHz,DMSO)δ8.91(dd,J=8.5,1.3Hz,1H),8.66(d,J=8.4Hz,1H),8.09(d,J =8.9Hz,1H),7.93(ddt,J=6.7,4.5,2.1Hz,3H),7.71–7.67(m,2H),7.66(d,J=3.9Hz,1H),7.63– 7.60(m,1H),7.53–7.50(m,2H),7.23(d,J=15.5Hz,1H),7.02(s,1H),6.86(d,J=8.4Hz,1H), 4.56(q,J=7.0Hz,2H),1.43(t,J=7.1Hz,3H).
图1为荧光探针SQM-NBD的高分辨质谱图,说明荧光探针SQM-NBD合成成功。
实施例2
母液配制
(1)配制荧光探针SQM-NBD母液:称取2.9mg SQM-NBD溶于5mL DMSO中,浓度为1mM/L。
(2)配制半胱氨酸(Cys)、高半胱氨酸(Hcy)、谷胱甘肽(GSH)母液:分别称取6.0mgCys、6.7mg Hcy和15.3mg GSH溶于50mL PBS缓冲液中,浓度为1mM/L。
实施例3
在5mL离心管中加入15μL实施例2配制的荧光探针SQM-NBD母液、15 μL DMSO和1.17mL PBS缓冲液,向混合溶液中分别加入1.8mL实施例2配制的Cys、Hcy和GSH母液,形成DMSO-PBS混合溶液(SQM-NBD浓度为5 μM/L,Fw=99%)。将离心管置于恒温水浴床中,在37℃下孵育30min。对收集溶液分别测定紫外吸收光谱,以测得的最大吸收波长为激发波长,分别测定Cys、Hcy和GSH溶液的荧光发射光谱。如图2中(A)所示,荧光探针 SQM-NBD与Cys/Hcy孵育后,荧光信号显著增强,对GSH几乎没有响应;如图2中(B)所示,荧光探针SQM-NBD与Cys和GSH共孵育后,荧光信号显著增强。同样采用荧光探针SQM-NBD与Hcy和GSH共孵育后,荧光信号依旧显著增强,说明本发明制备的荧光探针可以选择性检测Cys/Hcy,响应速度快。
实施例4
将Hela细胞置于培养瓶,在培养箱(37℃、5%CO2)中培养。24h后取出,在培养瓶中加入2mL胰酶消化液对细胞进行消化,待消化完成后加入2ml 培养基(含10%FBS的DEME培养基)终止消化。接着将细胞悬液离心,离心结束后弃去上液,再加入1mL培养基,用移液枪吹打使细胞重悬。将细胞悬液加入激光共聚焦皿中,置于5%CO2,37℃培养箱中过夜贴壁,弃去培养基,PBS缓冲液洗涤后加入2mL培养基。第一组加入10μL实施例2配制的 SQM-NBD母液共孵30min。第二组先用Cys(1mM/L)处理Hela细胞30 min,然后加入10μL实施例2配制的SQM-NBD母液,再共孵育30min。第三组首先用NEM处理Hela细胞30min,随后在细胞中加入10μL实施例2配制的SQM-NBD母液共孵育30min。用PBS缓冲液清洗三组处理后的细胞三次后,进行共聚焦成像。如图3所示,第一组呈现微弱的绿色荧光,表明SQM- NBD可以检测细胞中的内源性生物硫醇。第二组呈现强烈的绿色荧光,说明 SQM-NBD可以检测外源性Cys,且外源性Cys的浓度明显高于内源性的。第三组没有表现出荧光,表明NEM抑制了细胞内的生物硫醇,进一步印证了 SQM-NBD是与Cys/Hcy发生反应,可以检测内源性Cys/Hcy。因此,SQM-NBD能够检测活细胞中的内源性和外源性Cys/Hcy。
Claims (10)
2.一种权利要求1所述半胱氨酸/高半胱氨酸响应的AIE荧光探针的制备方法,其特征在于,包括如下步骤:
(1)将2-甲基喹啉和碘乙烷溶解于无水乙腈中,在惰性气体保护下回流反应,将反应混合物冷却至室温后,沉淀物减压抽滤,洗涤,真空干燥后得到化合物1;
(2)将化合物1与丙二腈溶解于乙醇中,然后加入乙醇钠反应,反应结束后,沉淀物减压抽滤,洗涤,真空干燥后得到化合物2;
(3)将4-羟基苯硼酸、5-溴-2-噻吩甲醛和四(三苯基膦)钯溶解在无水四氢呋喃中,加入碳酸钾水溶液,在惰性气体保护下回流反应,反应结束后,将反应混合物冷却至室温,萃取,减压蒸除溶剂,纯化后得到化合物3;
(4)将化合物2和化合物3溶解于无水乙腈中,再加入哌啶,在惰性气体氮气保护下回流反应,将反应混合物冷却至室温后,减压蒸除溶剂,纯化得到化合物SQM-OH。
(5)将SQM-OH与4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)溶解于无水二氯甲烷中,再加入三乙胺,在惰性气体保护下回流反应,反应结束后,将溶剂从混合物中蒸发,残余物纯化得到化合物探针SQM-NBD。
3.根据权利要求2所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针制备方法,其特征在于,步骤(1)将2-甲基喹啉和碘乙烷溶解于无水乙腈中,在惰性气体保护下于85℃~95℃反应回流12~15h,待反应结束后,将反应混合物冷却至室温后析出固体,减压抽滤去溶剂,得到滤饼用冷乙腈洗涤,真空干燥后,得到淡黄色固体,即化合物1。
4.根据权利要求2所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针制备方法,其特征在于,步骤(2)将化合物1与丙二腈溶解于乙醇溶液中,在-3℃~0℃下持续搅拌,然后加入乙醇钠,在-3℃~0℃下反应0.5~1h,转至室温下继续搅拌反应3-5h,反应完成后,沉淀物减压抽滤除去多余溶剂,得到滤饼用冷乙醇洗涤,真空干燥,得到黄色固体,即化合物2。
5.根据权利要求2所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针制备方法,其特征在于,步骤(3)将4-羟基苯硼酸、5-溴-2-噻吩甲醛和四(三苯基膦)钯溶解在无水四氢呋喃中,随后快速加入碳酸钾水溶液,在70~80℃、惰性气体保护下搅拌回流6~7h,将反应混合物冷却至室温,萃取,减压蒸除溶剂后,残余物通过柱层析纯化得化合物3。
6.根据权利要求2所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针制备方法,其特征在于,步骤(4)所述将化合物2和化合物3溶解在无水乙腈中,再加入哌啶,在70~85℃、惰性气体保护下反应回流7~8h,将反应混合物冷却至室温,减压蒸除溶剂后,残余物通过柱层析纯化得到化合物SQM-OH。
7.根据权利要求2所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针制备方法,其特征在于,步骤(5)所述将SQM-OH与NBD-Cl溶解于无水二氯甲烷中,再加入三乙胺,在25~30℃、惰性气体保护下反应回流9~12h,反应结束后,减压蒸除溶剂,残余物通过柱层析纯化得到荧光探针SQM-NBD。
8.一种利用权利要求1所述的半胱氨酸/高半胱氨酸响应的AIE荧光探针在半胱氨酸/高半胱氨酸的响应性检测和在细胞内源性外源性半胱氨酸/高半胱氨酸的成像中的应用。
9.根据权利要求8所述的AIE荧光探针在半胱氨酸/高半胱氨酸的响应性检测中的应用,其特征在于,所述响应检测的过程为:向含有探针的反应体系中分别加入半胱氨酸、高半胱氨酸和谷胱甘肽,将该反应溶液混合均匀后于36~38℃下孵育,孵育后测定紫外吸收和荧光发射光谱。
10.根据权利要求8所述的AIE荧光探针在细胞内源性外源性半胱氨酸/高半胱氨酸的成像中的应用,其特征在于,所述成像过程为:将Hela细胞于激光共聚焦皿中培养,将荧光探针加入皿中共孵育,用激光共聚焦显微镜进行成像。
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